This invention is generally related to DNA and RNA sequencing and, more particularly, to DNA and RNA sequencing by detecting individual nucleotides.
A world-wide effort is now in progress to analyze the base sequence in the human genome. The magnitude of this task is apparent, with 3.times.10.sup.9 bases in the human genome, and available base sequencing rates are about 200-500 bases per 1O-24 hour period. Considerable interest also exists in nucleic acid sequencing from non-human sources. Existing procedures are labor intensive and cost approximately $1 per base.
By way of example, Sanger et al., "DNA Sequencing with Chain-Terminating Inhibitors," proceedings of the National Academy of Science, USA 74, 5463-7 (1977) provide for sequencing 1-200 nucleotides from a priming site. Radioactive phosphorus is used in the primer extension to provide a marker. Enzymatic resynthesis coupled with chain terminating precursors are used to produce DNA fragments which terminate randomly at one of the four DNA bases adenine (A), cytosine (C), guanine (G), or thymine (T). The four sets of reaction products are separated electrophorectically in adjacent lanes of a polyacrylamide gel. The migration of the DNA fragments is visualized by the action of the radioactivity on a photographic film. Careful interpretation of the resulting band patterns is required for sequence analysis This process typically takes 1-3 days. Further, there are problems with band pile-ups in the gel, requiring further confirmatory sequencing.
In a related technique, A. M. Maxam and W. Gilbert, "A New Method for Sequencing DNA," proceedings of the National Academy of Science USA 74 560-564 (1977), teach a chemical method to break the DNA into four sets of random length fragments, each with a defined termination. Analysis of the fragments proceeds by electrophoresis as described above. The results obtained using this method are essentially the same as the "Sanger Method."
In another example, Smith et al., "Fluorescent Detection in Automated DNA Sequence Analysis," Nature 321, 674-679 (June 1986), teach a method for partial automation of DNA sequence analysis. Four fluorescent dyes are provided to individually label DNA primers. The Sanger method is used to produce four sets of DNA fragments which terminate at one of the four DNA bases with each set characterized by one of the four dyes. The four sets of reaction products, each containing many identical DNA fragments, are mixed together and placed on a polyacrylamide gel column. Laser excitation is then used to identify and characterize the migration bands of the labeled DNA fragments on the column where the observed spectral properties of the fluorescence are used to identify the terminal base on each fragment. Sequencing fragments of up to 400 bases has been reported. Data reliability can be a problem since it is difficult to uniquely discern the spectral identity of the fluorescent peaks.
These and other problems in the prior art are addressed by the present invention and an improved process is provided for rapid sequencing of DNA bases. As herein described, the present invention provides for the sequential detection of individual nucleotides cleaved from a single DNA or RNA fragment.
Accordingly it is an object of the present invention to provide an automated base sequence analysis for DNA and RNA.
Another object of the present invention is to process long strands of DNA or RNA, i.e., having thousands of bases.
One other object is to rapidly sequence and identify individual bases.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.